Dietary α-linolenic acid-rich flaxseed oil prevents against alcoholic hepatic steatosis via ameliorating lipid homeostasis at adipose tissue-liver axis in mice Meng Wang a, Xiao-Jing Zhang a, Kun Feng b, Chengwei He a, Peng Li a, Yuan-Jia Hu a, Huanxing Su a, Jian-Bo Wan a, * a State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao b Department of Bioengineering, Zunyi Medical College, Zhuhai Campus, Zhuhai, Guangdong, China *Corresponding author Dr. Jian-Bo Wan, Room 6034, Building N22, Avenida da Universidade University of Macau, Taipa, Macao. Tel: +853-8822 4680; E-mail: jbwan@umac.mo
Fatty acids % of total fatty acids Two-way ANOVA Common name Symbol PF/CO AF/CO PF/FO AF/FO Ethanol Fat Interaction Myristic acid 14:0 0.99 ± 0.06 1.15 ± 0.21 1.09 ± 0.19 0.88 ± 0.07 0.73 0.23 <0.05 Palmitic acid 16:0 19.1 ± 0.5 20.0 ± 0.9 17.8 ± 1.9 17.1 ± 1.0 0.83 <0.05 0.17 Palmitoleic acid 16:1 n-7 4.77 ± 0.82 3.41 ± 1.19 4.26 ± 0.99 3.02 ± 0.49 <0.05 0.30 0.88 Stearic acid 18:0 2.51 ± 0.39 3.98 ± 0.94 2.93 ± 0.48 4.59 ± 1.10 <0.05 0.24 0.83 Oleic acid 18:1 n-9 31.8 ± 0.9 a 30.0 ± 1.1 a, b 28.4 ± 2.7 b 27.5 ± 1.0 b 0.14 <0.001 0.05 Linoleic acid 18:2 n-6 38.0 ± 0.7 38.8 ± 1.7 26.9 ± 0.7 26.7 ± 1.7 0.67 <0.001 0.43 α-linolenic acid 18:3 n-3 1.61 ± 0.31 1.52 ± 0.50 19.6 ± 2.3 19.5 ± 2.1 0.90 <0.001 1.00 Dihomo-γ-linolenic acid 20:3 n-6 0.24 ± 0.02 0.27 ± 0.02 0.16 ± 0.02 0.16 ± 0.01 <0.05 <0.001 0.15 Arachidonic acid 20:4 n-6 0.44 ± 0.08 0.45 ± 0.05 0.21 ± 0.09 0.20±0.03 0.91 <0.001 0.68 Eicosapentaenoic acid 20:5 n-3 0.09 ± 0.03 0.08 ± 0.02 0.23 ± 0.12 0.19 ± 0.03 0.33 <0.05 0.65 Docosapentaenoic acid 22:5 n-3 0.04 ± 0.01 0.04 ± 0.01 0.13 ± 0.04 0.10 ± 0.05 0.55 <0.05 0.55 Docosahexaenoic acid 22:6 n-3 0.15 ± 0.02 0.22±0.05 0.21 ± 0.09 0.17 ± 0.04 0.55 0.10 0.09 SFAs 22.6 ± 0.9 25.1 ± 1.8 22.4 ± 3.2 22.6 ± 1.9 0.19 0.18 0.25 MUFAs 36.6 ± 0.9 a 33.4 ± 0.6 b 31.2 ± 1.1 c 30.5 ± 1.2 c <0.05 <0.001 <0.05 n-3 PUFAs 2.16 ± 0.65 1.90 ± 0.56 20.1 ± 2.4 19.9 ± 2.0 0.77 <0.001 0.98 n-6 PUFAs 38.7 ± 0.7 39.6 ± 1.7 27.2 ± 0.8 27.0 ± 1.8 0.61 <0.001 0.43 Total PUFAs 40.8 ± 1.2 41.5 ±1.4 46.4 ±2.2 46.9 ± 1.9 0.50 <0.001 0.93 n-6 PUFAs /n-3 PUFAs 16.6 ± 3.0 19.6 ± 5.2 1.43 ± 0.12 1.38 ± 0.21 0.32 <0.001 0.31 Supplementary Table S1. Fatty acid composition (%) of adipose tissues obtained from C57BL/6 mice pair-fed a control or an ethanol liquid diet with corn oil and flaxseed oil Values are expressed as means ± SD (n=4~6). Labeled means in a row without a common letter differ, P < 0.05. AF/CO, alcohol-fed with corn oil;
AF/FO, alcohol-fed with flaxseed oil; PF/CO, pair-fed with corn oil; PF/FO, pair-fed with flaxseed oil. SFAs value was calculated from (14:0 + 16:0 + 18:0); MUFAs value was calculated from (16:1 + 18:1); n-6 PUFAs was calculated from (18:2 n-6 + 20:3 n-6 + 20:4 n-6); n-3 PUFAs was calculated from (18:3 n-3 + 20:5 n-3 + 22:5 n-3 + 22:6 n-3); n-6 PUFAs /n-3 PUFAs means the ratio of n-6 PUFA to n-3 PUFA.
Ingredients PF/CO AF/CO PF/FO AF/FO Casein, g/l 41.4 41.4 41.4 41.4 L-cystine, g/l 0.50 0.50 0.50 0.50 D,L-Methionine, g/l 0.30 0.30 0.30 0.30 Cellulose, g/l 10.0 10.0 10.0 10.0 Maltose Dextrin, g/l 115 44.8 115 44.8 Corn Oil, g/l 39.6 39.6 - - Flaxseed Oil, g/l - - 39.6 39.6 Mineral Mix (AIN93G-MX), g/l 8.8 8.8 8.8 8.8 Vitamin Mix (AIN-93-VX), g/l 2.5 2.5 2.5 2.5 Choline Bitartrate, g/l 0.53 0.53 0.53 0.53 Vitamin E Acetate, g/l 0.20 0.20 0.20 0.20 95% Ethanol (v/v), ml/l - 52.6-52.6 Supplementary Table S2. Compositions of the modified Lieber-DeCarli liquid diets. AF/CO, alcohol-fed with corn oil; AF/FO, alcohol-fed with flaxseed oil; PF/CO, pair-fed with corn oil; PF/FO, pair-fed with flaxseed oil.
Fatty acids % of total fatty acids Common name Symbol Corn Oil Flaxseed Oil Lauric acid 12:0 0.2 0.2 Myristic acid 14:0 0.9 0.7 Palmitic acid 16:0 16.8 8.6 Palmitoleic acid 16:1 n-7 0.4 0.3 Stearic acid 18:0 4.1 5.4 Oleic acid 18:1 n-9 25.7 20.2 Linoleic acid (LA) 18:2 n-6 51.0 14.3 α-linolenicacid(ala) 18:3 n-3 1.0 50.3 SFAs 21.8 14.9 MUFAs 26.1 20.4 Total PUFAs 52.0 64.7 n-6 PUFAs/n-3 PUFAs 50.0 0.3 Supplementary Table S3. Fatty acid composition (%) of dietary fats contained in liquid diets. The relative content of each fatty acid was quantified by normalization of the peak areas as the percentages of total fatty acids; MUFAs, monounsaturated fatty acids; SFAs, saturated fatty acids; PUFAs, polyunsaturated fatty acids. n-6 PUFAs/n-3 PUFAs means the ratio of n-6 PUFAs to n-3 PUFAs.
Gene Full name GeneBank Primer sequences accession No. (forward/reverse) Cd36 Fatty acid translocase NM_001159556 5 -ATGGGCTGTGATCGGAACTG-3 5 -TTTGCCACGTCATCTGGGTTT-3 Fatp1 Fatty acid transport protein 1 NM_011977 5 -CGCTTTCTGCGTATCGTCTG-3 5 -GATGCACGGGATCGTGTCT-3 Lpl Lipoprotein lipase NM_008509 5 -TTGCCCTAAGGACCCCTGAA-3 5 -TTGAAGTGGCAGTTAGACACAG-3 Vldl-r Very low-density lipoprotein receptor MMU06670 5 -GGCAGCAGGCAATCGAATG-3 5 -GGGCTCGTCACTCCAGTCT-3 Fatp2 Fatty acid transport protein 2 NM_011978 5 -TCCTCCAAGATGTGCGGTACT-3 5 -TAGGTGAGCGTCTCGTCTCG-3 Fatp5 Fatty acid transport protein 5 NM_009512 5 -TCTATGGCCTAAAGTTCAGGCG-3 5 -CTTGCCGCTCTAAAGCATCC-3 Gpat Glycerol-3-phosphate acyltransferase NM_008149 5 -ACAGTTGGCACAATAGACGTTT-3 5 -CCTTCCATTTCAGTGTTGCAGA-3 Dgat1 Diacylglycerol O-acyltransferase 1 NM_010046 5 -TCCGTCCAGGGTGGTAGTG-3 5 -TGAACAAAGAATCTTGCAGACGA-3 Dgat2 Diacylglycerol O-acyltransferase 2 NM_026384 5 -GCGCTACTTCCGAGACTACTT-3 5 -GGGCCTTATGCCAGGAAACT-3 β-actin Beta-actin NM_007393 5 -GGCTGTATTCCCCTCCATCG-3 5 -CCAGTTGGTAACAATGCCATGT-3 Supplementary Table S4. A list of genes and their corresponding primers used in the quantitative RT-PCR analysis.
Primary Molecular Full name antibody Weight Maker ATGL Adipose triglyceride lipase 54 kda Cell Signaling p-hsl(ser 660 ) Phospho-hormone-sensitive lipase 81 kda Cell Signaling HSL Hormone-sensitive lipase 81 kda Cell Signaling p-ire1α Phospho-inositol-requiring enzyme 1 (Ser 724 ) alpha 110 kda Abcam IRE1α Inositol-requiring enzyme 1 alpha 110 kda Abcam p-eif2α Phospho-eukaryotic translation (Ser 51 ) initiation factor 2 subunit alpha 38 kda Bioworld Technology EIF2α Eukaryotic translation initiation factor 2 subunit alpha 38 kda Bioworld Technology PPARG Peroxisome proliferator-activated receptor- gamma 53,57 kda Cell Signaling CD36 Fatty acid translocase 88 kda Santa Cruz Biotechnology FATP5 Fatty acid transport protein 5 75 kda Santa Cruz Biotechnology MTTP Microsomal triglyceride transfer protein 97 kda Santa Cruz Biotechnology ADIPOR2 Adiponectin receptor 2 44 kda Santa Cruz Biotechnology p-ampk Phospho-AMP-activated protein (Thr 172 ) kinase 62 kda Cell Signaling AMPK AMP-activated protein kinase 62 kda Cell Signaling p-acc (Ser 79 ) Phospho-acetyl-CoA carboxylase 280 kda Cell Signaling ACC acetyl-coa carboxylase 280 kda Cell Signaling β-actin BETA-ACTIN 42 kda Cell Signaling Supplementary Table S5. Primary antibodies used in the immunoblot analysis.